Disease Information: The parasitic protozoan Trypanosomabrucei is the causative agent of human African trypanosomiasis (sleeping sickness) and nagana. Sleeping sickness is caused by infection with protozoan parasites. The parasite is transmitted to humans through the bite of the tsetse fly. The disease was brought under control in the mid-1960s through intensive control programmes, but re-emerged due to weakened surveillance, reaching epidemic proportions in several regions by 1970. Current drug therapies have limited efficacy, high toxicity and/or are continually hampered by the appearance of resistance. Antimicrobial peptides have recently attracted attention as potential parasiticidal compounds.
http://www.who.int/trypanosomiasis_african/news/The-world-gears-up-to-eliminate-HAT-2020/en/ Enzyme Information: Dihydroorotate dehydrogenase EC 1.3.5.2 (previously 1.3.99.11) is a flavoenzyme that catalyzes the oxidation of (//S//)-dihydroorotate to orotate with the reduction of FMN to FMNH2, a key reaction in the de novo pyrimidine biosynthetic pathway ([Fagan06] and reviewed in [Loffler15]). It is a Class 2 enzyme that utilizes a ubiquinone as the electron transfer quinone and is coupled to the mitochondrial respiratory chain [Fang13a, Hines86]. In mammals the enzyme is an integral membrane protein found on the outer surface of the mitochondrial inner membrane, exposed to the intermembrane space [Rawls00]. It contains a serine residue that functions as the active site base during catalysis [Jiang00a]. Inhibitors of human dihydroorotate dehydrogenase are of interest in drug development.
Comment:
The reaction, which takes place in the cytosol, is the only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Molecular oxygen can replace fumarate in vitro. Other class 1 dihydroorotate dehydrogenases use either NAD(+) (EC 1.3.1.14) or NADP(+) (EC 1.3.1.15) as electron acceptor. The membrane bound class 2 dihydroorotate dehydrogenase (EC 1.3.5.2) uses quinone as electron acceptor. Formerly Ec 1.3.3.1.
https://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/enzymes/GetPage.pl?ec_number=1.3.98.1 Essentiality: Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection.
Assay and Expression Information: Dihydroorotate oxidases have been highly purified from the parasitic protozoa Crithidia fasciculata and Trypanosoma brucei. The Crithidia enzyme was purified 4200-fold from a crude soluble protein extract in four steps. The protein is a dimer as judged from the native (Mr 60 000) and subunit (Mr 32 700) molecular weights. The purified enzyme exhibits a characteristic flavin electronic spectrum, and each mole of native dimer contains 1.0 mol of tightly bound flavin mononucleotide. Under anaerobic conditions, the flavin chromophore is reduced upon addition of L-dihydroorotate. In air-saturated buffer, the enzyme catalyzes the conversion of L-dihydroorotate to orotate with concomitant reduction of equimolar amounts of molecular oxygen to hydrogen peroxide.
NCBI Gene # or RefSeq#: NT_165288.1
Protein ID: 2B4G
E.C Number: 1.3.98.1
Organism: Trypanosoma brucei brucei
Etiologic Risk Group: n/a
Disease Information:
The parasitic protozoan Trypanosomabrucei is the causative agent of human African trypanosomiasis (sleeping sickness) and nagana.
Sleeping sickness is caused by infection with protozoan parasites. The parasite is transmitted to humans through the bite of the tsetse fly.
The disease was brought under control in the mid-1960s through intensive control programmes, but re-emerged due to weakened surveillance, reaching epidemic proportions in several regions by 1970. Current drug therapies have limited efficacy, high toxicity and/or are continually hampered by the appearance of resistance. Antimicrobial peptides have recently attracted attention as potential parasiticidal compounds.
https://www.ncbi.nlm.nih.gov/pubmed/29649664
http://www.who.int/trypanosomiasis_african/news/The-world-gears-up-to-eliminate-HAT-2020/en/
Enzyme Information:
Dihydroorotate dehydrogenase EC 1.3.5.2 (previously 1.3.99.11) is a flavoenzyme that catalyzes the oxidation of (//S//)-dihydroorotate to orotate with the reduction of FMN to FMNH2, a key reaction in the de novo pyrimidine biosynthetic pathway ([Fagan06] and reviewed in [Loffler15]). It is a Class 2 enzyme that utilizes a ubiquinone as the electron transfer quinone and is coupled to the mitochondrial respiratory chain [Fang13a, Hines86]. In mammals the enzyme is an integral membrane protein found on the outer surface of the mitochondrial inner membrane, exposed to the intermembrane space [Rawls00]. It contains a serine residue that functions as the active site base during catalysis [Jiang00a].
Inhibitors of human dihydroorotate dehydrogenase are of interest in drug development.
https://biocyc.org/gene?orgid=HUMAN&id=HS02434-MONOMER
Comment:
The reaction, which takes place in the cytosol, is the only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Molecular oxygen can replace fumarate in vitro. Other class 1 dihydroorotate dehydrogenases use either NAD(+) (EC 1.3.1.14) or NADP(+) (EC 1.3.1.15) as electron acceptor. The membrane bound class 2 dihydroorotate dehydrogenase (EC 1.3.5.2) uses quinone as electron acceptor. Formerly Ec 1.3.3.1.
https://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/enzymes/GetPage.pl?ec_number=1.3.98.1
Essentiality:
Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection.
https://www.rcsb.org/structure/2B4G
BRENDA Page: http://www.brenda-enzymes.org/enzyme.php?ecno=1.3.98.1&Suchword=&reference=&UniProtAcc=&organism%5B%5D=Trypanosoma+brucei+brucei
Enzyme Image:
TDR Target page: http://tdrtargets.org/targets/view?gene_id=13795
Associated Compounds and Druggability:
Druggability Index: 0.9
Assay and Expression Information:
Dihydroorotate oxidases have been highly purified from the parasitic protozoa Crithidia fasciculata and Trypanosoma brucei. The Crithidia enzyme was purified 4200-fold from a crude soluble protein extract in four steps. The protein is a dimer as judged from the native (Mr 60 000) and subunit (Mr 32 700) molecular weights. The purified enzyme exhibits a characteristic flavin electronic spectrum, and each mole of native dimer contains 1.0 mol of tightly bound flavin mononucleotide. Under anaerobic conditions, the flavin chromophore is reduced upon addition of L-dihydroorotate. In air-saturated buffer, the enzyme catalyzes the conversion of L-dihydroorotate to orotate with concomitant reduction of equimolar amounts of molecular oxygen to hydrogen peroxide.
http://www.brenda-enzymes.org/literature.php?e=1.3.98.1&r=390910
There are no transmembrane regions
TMpred graph Image (https://embnet.vital-it.ch/cgi-bin/TMPRED_form_parser)
Gene Available: Yes https://www.atcc.org/Search_Results.aspx?dsNav=Ntk:PrimarySearch%7cTrypanosoma+brucei+brucei%7c3%7c,Ny:True,Ro:0,N:1000552&searchTerms=Trypanosoma+brucei+brucei&redir=1
CDS Gene Sequence:
Trypanosama brucei brucei Protein Sequence (https://www.ncbi.nlm.nih.gov/protein/XP_828627.1):
dihydrooroate oxidase Gene Sequence (Hono Sapiens):
dihydrooroate oxidase Protein Sequence (https://www.ncbi.nlm.nih.gov/gene/1723):